The present invention relates to methods of modulating levels/activity of 70 kilodalton heat-shock protein (HSP70) family members, for regulating vesicular shedding of complement, for regulating complement-mediated cytotoxicity, and for treating diseases which are associated with pathological cells and are treatable via complement-mediated cytolysis of such cells, and/or which are associated with pathological complement-mediated cytolysis; and to articles of manufacture which comprise compounds for practicing such methods. The present invention more particularly relates to methods of decreasing levels/activity of mortalin for decreasing vesicular shedding of complement, for increasing complement-mediated cytolysis of pathological cells, and for treating diseases, such as tumoral, infectious, autoimmune and transplantation-related diseases, which are associated with such cells, and are treatable via complement-mediated cytolysis of, such cells, where such methods are effected using: substantially cell membrane-impermeable compounds; and/or compounds for decreasing levels/activity of mortalin in combination with compounds for increasing association of complement with pathological cells. The present invention further particularly relates to methods of increasing levels/activity of mortalin for decreasing vesicular shedding of complement, for decreasing pathological complement-mediated cytotoxicity, and for treating diseases associated with such pathological cytotoxicity, such as autoimmune, immune complex, and transplantation-related diseases; and to articles of manufacture which comprise compounds for practicing such methods.
Diseases such as tumoral, infectious, autoimmune and transplantation-related diseases, which are associated with pathological cells and are treatable via complement-mediated cytolysis of such cells represent numerous highly debilitating and/or lethal diseases for which no optimal therapy exists. Similarly, diseases associated with pathological complement-mediated cytotoxicity, such as autoimmune, immune-complex and transplantation-related diseases, also represent numerous highly debilitating and/or lethal diseases for which no optimal therapy exists. There is therefore a long-felt and urgent need in the art for novel and maximally effective methods and therapeutic agents for treating such diseases.
The complement system consists of more than twenty blood plasma proteins that cooperate with other sections of the innate and acquired immune systems in clearance of pathogenic organisms, immune complexes and apoptotic cells (Walport, M. J. 2001. N Engl J Med 344:1058). The complement activation cascade culminates in formation of the membrane attack complex (MAC), made of complement C5b, C6, C7, C8 and C9 proteins (termed “C5b-9”), and its insertion into the plasma membrane of target cells (Muller-Eberhard, H. J. 1986. Annu Rev Immunol 4:503). Membrane insertion begins when C5b-7 forms, is enhanced upon formation of C5b-8 complex and is maximal upon binding and oligomerization of C9 and formation of a transmembrane, cylinder-shape polyC9 complex attached to C5b-8. At supralytic doses, MAC normally functions to induce rapid cell death by necrosis (Koski, C. L. et al.,1983. Proc Natl Acad Sci U S A 80:3816) or apoptosis (Cragg, M. S. et al., 2000. Cell Death Differ 7:48). At low, sublytic doses, MAC acts as a potent stimulator of numerous cellular activities (for review see Bohana-Kashtan, O. et al., 2004. Mol Immunol 41:583). Treatment with sublytic MAC has been shown to transduce either anti-necrotic (Reiter, Y. et al., 1992. Eur J Immunol 22:1207) or anti-apoptotic (Dashiell, S. M. et al., 2000. Glia 30:187) signals into various cells.
As a means of protection from complement, nucleated cells can remove the MAC from their plasma membrane by endocytosis or vesiculation (Sims, P. J. and Wiedmer, T. 1986. Blood 68:556; Morgan, B. P. et al., 1987. J Immunol 138:246; Carney, D. F. et al., 1985. J Immunol 134:1804) or proteolytic fragmentation. Physical removal of MAC by vesiculation has been demonstrated in several cell types including neutrophils, oligodendrocytes and platelets, and in the tumor cell lines U937 and K562 (Sims, P. J. and Wiedmer, T. 1986. Blood 68:556; Scolding, N. J. et al., 1989. Nature 339:620; Morgan, B. P. et al., 1986. J Immunol 136:3402; Morgan, B. P. 1992. Curr Top Microbiol Immunol 178:115). The shed vesicles have a high content of cholesterol and diacylglycerol (Stein, J. M. and Luzio, J. P. 1991. Biochem J. 274 (Pt 2):381) and are loaded with MAC and C9, suggesting a selective sorting on the cell surface prior to shedding. To date, little is known about the molecular mechanism responsible for MAC vesiculation. Extracellular Ca2+ has been suggested to play a role in elimination of terminal complement complexes (Carney DF. et al., 1986. Elimination of terminal complement complexes in the plasma membrane of nucleated cells: influence of extracellular Ca2+ and association with cellular Ca2+. J Immunol. 137:263-70). Various proteins capable of regulating complement activity are known. In particular, three membrane complement regulatory proteins (mCRPs) inhibit complement activation: decay accelerating factor (DAF, CD55), membrane cofactor protein (MCP, CD46) and CD59.
Removal of complement from nucleated cells may be associated with disease pathogenesis. For example, MAC removal has been shown to protect cancer cells from complement-mediated cytotoxicity. Lysis of tumor cells by homologous complement is inefficient primarily due to their capacity to subvert complement binding and damage. In general, tumor cell protective mechanisms may be divided into intrinsic and induced mechanisms. Intrinsic mechanisms determine the basal resistance of the tumor cells to homologous complement, and the induced protective mechanisms represent the capacity of the tumor cell to react to various external stimuli (for example, cytokines, toxins, hormones as well as an ongoing complement activation) and to increase its level of protection from complement. Membrane complement regulatory proteins are over-expressed on the surface of cancer cells, and render them resistant to autologous complement (Fishelson Z. et al., 2003. Obstacles to cancer immunotherapy: expression of membrane complement regulatory proteins (mCRPs) in tumors. Mol Immunol. 40:109-23). Neutralization of mCRPs with blocking antibodies sensitizes both human leukemic and carcinoma cells to lysis by human complement. Thus, the capacity of cells to shed MAC so as to avoid cytotoxicity is problematic for disease treatment approaches involving antibody-mediated cytolysis of pathological cells. In recent years, new monoclonal antibodies have been designed to target and kill tumor cells. This era of targeted therapy has brought to the clinic a handful of monoclonal antibodies, including Rituxan (rituximab), designed for relapsed or refractory CD20-positive non-Hodgkin B-cell lymphoma, Herceptin (trastuzumab) for breast tumors overexpressing the human epidermal growth factor receptor 2 (HER-2) and others. While such disease treatments have only yielded partial benefits, there is nevertheless great interest in antibody-based therapeutics for hematopoietic malignant neoplasms and solid tumors, due to the inefficiency and harmful side-effects of conventional cancer treatment approaches which involve chemo- and radio-therapy. Various studies have investigated the role of complement-mediated lysis in antibody-mediated cancer therapy in an attempt to elucidate means of achieving therapeutic improvement via such treatment. By introducing human IgGI heavy and light chain domains, the antiproliferative properties of a precursor murine monoclonal anti-p185HER antibody to Herceptin were extended by its capacity to induce antibody-dependent cell-mediated cytotoxicity (ADCC). It has been shown that complement activation on various HER-2 positive tumor cell lines upon sensitization with the humanized anti-p185HER antibody leads to opsonization of the tumor cells with C3b. However, complement-mediated tumor cell lysis became only possible upon neutralization of mCRP. Part of the antitumor effect of rituximab has been ascribed to its capacity to bind C1q, activate complement and eventually kill the cells.
Targeting of proteins capable of regulating complement has been suggested for tumor therapy (Harris, C. et al., 1997. Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59. Clin Exp Immunol 107:364; Blok, V. T. et al., 1998. A bispecific monoclonal antibody directed against both the membrane-bound complement regulator CD55 and the renal tumor-associated antigen G250 enhances C3 deposition and tumor cell lysis by complement. J Immunol 160:3437; Gelderman, K. A. et al., 2002. The inhibitory effect of CD46, CD55, and CD59 on complement activation after immunotherapeutic treatment of cervical carcinoma cells with monoclonal antibodies or bispecific monoclonal antibodies. Lab Invest 82:483; Gelderman, K. A. et al., 2004. Tumor-specific inhibition of membrane-bound complement regulatory protein Crry with bispecific monoclonal antibodies prevents tumor outgrowth in a rat colorectal cancer lung metastases model. Cancer Res 64:4366.). One possible approach is the construction of bispecific monoclonal antibodies consisting of one Fab moiety directed to a tumor specific antigen and another directed to an mCRP. Tumor-directed bispecific antibodies with an anti-mCRP moiety would enable specific targeting of complement regulators on tumor cells without impairment of healthy tissue. Proof of concept that a bispecific antibody directed against an mCRP may be protective against malignant cells has been achieved in-vitro. Several forms of bispecific antibodies have been generated to link tumor cells more effectively to immune effector cells and some of them are already in clinical trials. For example, a phase II study showed that the bispecific antibody MDX-H210 (anti-HER2/anti-CD64) together with GM-CSF is therapeutically active against hormone refractory HER2+ prostate cancer.
Mechanisms protecting cells from heat-shock and from complement share some resemblance. For example, both of these shock responses depend on de-novo protein synthesis, exhibit similar functional kinetics, and studies have suggested a role for members of the 70 kilodalton heat shock protein (HSP70) family proteins in regulation of complement-mediated cytolysis (Fishelson Z. et al., 2001. Contribution of heat shock proteins to cell protection from complement-mediated lysis. Int Immunol. 13:983-991).
Mortalin, also known as GRP75, PBP74, mitochondrial HSP75 and mot-2, is a member of the HSP70 family (GeneCard #GC05M137967). This protein has been assigned multiple functions including stress response (Carette, J. et al., 2002. Int J Radiat Biol 78:183), glucose regulation, p53 inactivation, control of cell proliferation, differentiation, tumorigenesis and mitochondrial import (reviewed in Wadhwa, R. et al., 2002. Cell Stress Chaperones 7:309; Voisine, C. et al., 1999. Cell 97:565). Mortalin has been mainly described inside cells, in mitochondria and several other cytoplasmic locations such as endoplasmic reticulum and cytoplasmic vesicles (Ran, Q. et al., 2000. Biochem Biophys Res Commun 275:174). Mortalin is ubiquitously and constitutively expressed in normal tissues, and has been shown to be displayed on the surface of mouse B-cells and macrophages (VanBuskirk, A. M. et al., 1991. J Immunol 146:500). Its expression level is upregulated in some tumors, such as neuroblastoma, lung adenocarcinoma, leukemia and ovarian cancer cells (Takano, S. et al., 1997. Exp Cell Res 237:38; Dundas, SR. et al., 2004. J Pathol 205:74; Shin, B. K. et al., 2003. J Biol Chem 278:7607), as well as during infection and inflammation (Kirmanoglou, K. et al., 2004. Basic Res Cardiol 99:404; Johannesen, J. et al., 2004. Is mortalin a candidate gene for T1DM ? Autoimmunity 37:423). Overexpression of mortalin in normal cells considerably extends their lifespan (Kaul, S. C. et al., 2003. Exp Cell Res 286:96), while reduction of mortalin levels in immortalized cells causes growth arrest (Wadhwa, R. et al., 2004. J Gene Med 6:439; Wadhwa et al., 1994. Cellular mortality to immortalization: mortalin. Cell Struct Funct. 19:1-10). In view of the expression of mortalin in cancers, the use of this protein as therapeutic target has been proposed (Wadhwa R. et al., 2002. Mortalin: a potential candidate for biotechnology and biomedicine. Histol Histopathol. 17:1173-7).
Thus, in view of the possible role of HSP70 family proteins in mediating protection from complement-mediated cytotoxicity, and in view of the overexpression of such proteins in pathological cells susceptible to elimination via such cytotoxicity, suitable modulation of levels/activity of such proteins may represent a potentially optimal strategy for treating diseases associated with pathological complement-mediated cytotoxicity, and/or associated with pathological cells and treatable via complement-mediated cytolysis of such cells.
Several prior art approaches have been proposed involving decreasing levels/activity of HSP70 family proteins, such as mortalin, for treating diseases associated with pathological cells and treatable via complement-mediated cytolysis of such cells.
One approach involves administration of the mortalin inhibitor MKT-077 (formerly FJ-776) for treatment of cancers characterized by wild-type p53 (Wadhwa R. et al., 2000. Cancer Research 60, 6818-6821), chemo-resistant solid tumors (Propper D. J. et al., 1999. Phase I trial of the selective mitochondrial toxin MKT077 in chemo-resistant solid tumours. Ann. Oncol., 10: 923-927), untreatable/treatment-refractory solid tumors (Britten C. D. et al, 2000. A Phase I and pharmacokinetic study of the mitochondrial-specific rhodacyanine dye analog MKT 077. Clin. Cancer Res., 6: 42-49), or solid tumors of various lineages (Wadhwa R. et al., 2002. Cancer Res. 62:4434-8). This approach however, was found to be non-practicable due to MKT-077 causing irreversible kidney damage in human patients (Propper D. J. et al., 1999. Ann. Oncol., 10: 923-927), and was ineffective or suboptimally effective when used to treat cancer patients.
Another approach involves expression of mortalin anti-sense RNA in cancer cells for treatment of cancers characterized by compromised p53 and pRB functions and telomerase activity (Wadhwa R. et al., 2004. Reduction in mortalin level by its antisense expression causes senescence-like growth arrest in human immortalized cells. J Gene Med. 6:439-44). This approach however, has the significant disadvantages of being only potentially relevant to cancers characterized by compromised p53 and pRB functions and telomerase activity; and of having been investigated in synthetically immortalized cells; of not having been investigated in-vivo.
A further approach involves expression of conventional or RNA-helicase-coupled hammerhead ribozymes for treatment of cancers (Wadhwa R. et al., 2003. Targeting mortalin using conventional and RNA-helicase-coupled hammerhead ribozymes. EMBO Rep. 4:595-601). This approach however, has the significant disadvantages of being only potentially relevant to synthetically immortalized cells; and of not having been investigated in-vivo.
An additional approach suggests using mortalin as molecular target for treatment of hepatitis C virus-related hepatocellular carcinoma (Takashima M. et al., 2003. Proteomics. 3:2487-93). This approach, however, has the significant disadvantages of not having been experimentally attempted, and of being limited to potential treatment of hepatitis C virus-related hepatocellular carcinoma.
Yet a further approach suggests employing inhibition of HSC70 with deoxyspergualin to increase the sensitivity of K562 human erythroleukemia cells to complement-mediated lysis (Fishelson Z. et al., 2001. Contribution of heat shock proteins to cell protection from complement-mediated lysis. Int Immunol. 13:983-991). This approach, however, has the significant disadvantages of not having been experimentally attempted in-vivo nor against primary tumor cells.
Various prior art approaches have been proposed involving increasing levels/activity of HSP70 family proteins for treating diseases associated with pathological complement-mediated cytotoxicity.
One approach involves upregulating HSP70 synthesis using the amino acid analogue L-azetidine-2-carboxylic acid for defending cells against complement-mediated lysis (Fishelson Z. et al., 2001. Contribution of heat shock proteins to cell protection from complement-mediated lysis. Int Immunol. 13:983-91). This approach, however, has the significant disadvantages of not having been experimentally attempted in-vivo, nor against affected cells of a disease associated with pathological complement-mediated cytotoxicity.
A further approach suggests upregulating HSC70 synthesis, via treatment with ethanol, butanol or hemin, to protect cells from complement-mediated cytolysis (Fishelson Z. et al., 2001. Contribution of heat shock proteins to cell protection from complement-mediated lysis. Int Immunol. 13:983-991). This approach, however, also has the significant disadvantages of not having been experimentally attempted in-vivo nor against affected cells of a disease associated with pathological complement-mediated cytotoxicity.
Another approach involves using HSP70 to inhibit complement activation for treating xenograft rejection (Gralinski M R. et al., 1996. Am J Physiol. 271:H571-8). This approach, however, has the significant disadvantage of not having been attempted experimentally.
Critically, no prior art approach involving modulation of HSP70 family member proteins for disease treatment has demonstrated satisfactory/optimal therapeutic effectiveness.
Thus, all prior art approaches have failed to provide an adequate solution for using modulation of levels/activity of HSP70 family member proteins, such as mortalin, for treatment of diseases associated with pathological complement-mediated cytotoxicity, and/or associated with pathological cells and treatable via complement-mediated cytotoxicity of such cells.
There is thus a widely recognized need for, and it would be highly advantageous to have novel, maximally effective methods of treating diseases via modulation of levels/activity of HSP70 family proteins, such as mortalin, devoid of the above limitation.